Subcellular localization of proHNP processing.

<p>(A) PLB-985 cells were pelleted and disrupted by nitrogen cavitation. After low speed centrifugation, the cavitate was divided in a postnuclear pellet (P₁) consisting of nuclei and unbroken cells and a post-nuclear supernatant (S₁) containing cytosol, organelles (including granules), and cell membranes. S₁ was underlaid with a two-layer 1.05/1.07 PBS/Percoll density gradient and centrifuged at 37.000<i>g</i> for 30 minutes. Fractions were collected from the bottom of the gradient. Percoll was removed from fractions by ultracentrifugation. (B) Fractions were subjected to Western blotting for HNP, proHNPs, the endoplasmic reticulum (ER) marker calnexin, and the Golgi marker RCAS1. (C) PLB-985 cells were pulsed overnight in medium containing 100 μCi/mL ³⁵S-methionine/cysteine. Cells were pelleted and the supernatant used for isolation of ³⁵S-labelled proHNP by affinity chromatography with an antibody specific for proHNP. Radioactive fractions were pooled, dialyzed against PBS, and tested for proHNP by 16% SDS-Tricine-PAGE and fluorography. (D) ³⁵S-proHNP was incubated with subcellular fractions of PLB-985 for 15 hours at 37°C. Processing was tested by 16% SDS-Tricine-PAGE and fluorography.</p

Processing of proHNP-1 by MPRO cells transfected with human serine proteases.

<p>(A) MPRO cells and MPRO cells transfected with HNP-1 were spun onto slides, fixed, permeabilized, and immunocytochemically stained for IgG (top), HNPs (middle), and proHNPs (bottom). Bars represent 20 μm. (B) MPRO-HNP-1 cells were further transfected with the human neutrophil serine proteases neutrophil elastase (<i>ELANE</i>), cathepsin G (<i>CTSG</i>), and proteinase 3 (<i>PRTN3</i>). Cells were pulsed with ³⁵S-methionine/cysteine for 1 hour and chased overnight. Cell lysates and medium were immunoprecipitated with antibodies in the following order: anti-proHNP, and anti-HNP. Immunoprecipitates were pooled and analyzed by 16% SDS-Tricine-PAGE and fluorography using ³⁵S-proHNP and ³⁵S-HNP from the proHNP processing assay as controls. (C) Comparative quantification mRNA for <i>ELANE</i>, <i>CTSG</i>, and <i>PRTN3</i> was performed by real-time PCR. Figure depicts expression levels relative to cells electroporated human serine proteases. Bars represent means and lines represent standard deviation. (D) Activity of transfected human neutrophil elastase and proteinase 3 was asserted by lysis of transfected MPRO cells followed by spectrophotometry following degradation rate of methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide.</p

Papillon-Lefévre patients.

<p>Mutations of the Papillon-Lefévre patients. Nucleotides are numbered according to the coding DNA sequence (CDS).</p><p>Papillon-Lefévre patients.</p

Structure of preproHNP-1-3.

<p>Arrows indicate major sites of proteolytic cleavage. Positively and negatively charged amino acids are indicated in red and green, respectively. Lines indicate the disulphide linkage of cysteines (C; orange). HNP-3 is identical to HNP-1 except for having substituted alanine (A) at position 65 for aspartic acids (D).</p

Serine protease activity is not required for HNP processing <i>in vivo</i>.

<p>Peripheral blood neutrophils from two patients suffering from Papillon–Lefèvre syndrome (PLS). (A) Neutrophil elastase (NE) and proteinase 3 (PR3) activity was measured as absorbance (Abs) following degradation of methoxysuc-AAPV-p-nitroanilide. Assay from patient 1 is shown and is representative of both patients. (B) Cathepsin G (CG) activity was measured as absorbance following degradation of N-suc-AAPF-p-nitroanilide. Assay from patient 1 is shown and is representative of both patients. (C) Western blotting of NE, CG, and PR3 in two PLS patients and controls. (D) Western blotting of HNP and β-actin in two PLS patients and controls.</p

Additional file 3: Figure S2. of Impact of microRNA-130a on the neutrophil proteome

Expression of miR-130a following transfection with an anti-miR-130a-LNA or scrambled-LNA of the 32Dcl3 miR-130a clone for 48 h (left) and Kasumi-1 cells for 72 h (right) measured by real-time PCR. The large difference in the level of free miR-130a in the 32Dcl3 miR-130a clone compared to Kasumi-1 cells is presumably due to the former cells being more susceptible to transfection than the latter. Error bars represent SD between triplicate measurements in the real-time PCR experiment. (TIF 44 kb

Characterization of granulocytic bone marrow cells of transgenic HNP-1 mice deficient in C/EBP-ε.

<p>Murine bone marrow cells were extracted from wild type and transgenic HNP-1 mice with or without deficiency in C/EBP-ε. Erythrocytes and non-granulocytic cells removed by ammonium chloride-based lysis and by immunomagnetic sorting, respectively. Comparative quantification of mRNA for CCAAT/enhancer binding protein-ε (<i>Cebpe</i>), human neutrophil peptide-1 (<i>DEFA1</i>), myeloperoxidase (<i>Mpo</i>), 24p3 (<i>Lcn2</i>), lactoferrin (<i>Ltf</i>), cathelicidin antimicrobial peptide (<i>Camp</i>), matrix metalloproteinase-9 (<i>Mmp9</i>) was done by real-time PCR using <i>Gapdh</i> as normalizer. Figure depicts expression levels relative to HNP-1, Cebpe^+/+ mice. Error bars were calculated by Stratagene MxPro 4.1. Data are representative of two independent experiments.</p

Biosynthesis and Western blotting of proHNP and HNP in HNP-1 mice deficient in C/EBP-ε.

<p>Murine bone marrow (BM) cells from one wild type and transgenic HNP-1 mice with or without deficiency in C/EBP-ε were extracted. (A) Cells were pulsed with ³⁵S-methionine/cysteine for 1 hour and chased for 5 hours. Cell lysates and medium were immunoprecipitated with antibodies against proHNP and HNP. Immunoprecipitates were pooled and analyzed by 16% SDS-Tricine-PAGE and fluorography. (B) Non-granulocytic cells from BM of wild type mice, transgenic HNP-1 mice (HNP-1, Cebpe^+/+), and HNP-1 mice heterozygous or deficient for C/EBP-ε (HNP-1, Cebpe^+/− or Cebpe^-/-) were removed by ammonium chloride-based lysis and by immunomagnetic sorting. Western blotting was performed for HNP, proHNP, and GAPDH and is representative of two independent experiments. (C) BM cells from Cebpe^+/+ (blue) or Cebpe^-/-mice (orange) were immunomagnetically depleted of non-granulocytic cells and labeled with biotinylated rat anti-mouse CD11b antibody and analyzed by flow cytometry using biotinylated rat anti-mouse IgG2b as isotype control. Data are representative of four independent experiments.</p

Human and murine C/EBP-ε induces expression of HNP-1 in primary bone marrow (BM) cells from transgenic HNP-1 mice.

<p>Murine BM cells from seven transgenic HNP-1 mice were isolated and early granulocyte precursors isolated by density centrifugation on a discontinuous Percoll 1.072 gradient. Cells were retrovirally transduced with an empty expression vector (pMIG) or with a vector expressing either human or murine C/EBP-ε (pMIG-CEBPE or pMIG-Cebpe respectively). Cells were incubated for 48 hours. (A) Green fluorescent protein (GFP) was used as reporter gene in the vectors and transduction efficiency evaluated by flow cytometry. (B–G) Comparative quantification of mRNA for CCAAT/enhancer binding protein-ε (human <i>CEBPE</i> or murine <i>Cebpe</i>), human neutrophil peptide-1 (<i>DEFA1</i>), cathelicidin antimicrobial peptide (<i>Camp</i>), and lipocalin-2 (<i>Lcn2</i>) was done by real-time PCR using <i>Gapdh</i> as normalizer. Error bars depict standard deviation. (B, E–G) Levels are shown as fold induction by either murine <i>Cebpe</i> (mCebpe) or human <i>CEBPE</i> (hCEBPE) compared to levels from negative control transduction (pMIG). (C) Relative quantification of human <i>CEBPE</i> in murine bone marrow cells from four transgenic HNP-1 mice transduced with control vector (pMIG) or human <i>CEBPE</i>. (D) Expression of murine <i>Cebpe</i> in <i>Cebpe</i> transduced cells were compared to human <i>CEBPE</i> in <i>CEBPE</i> transduced cells by comparing Delta Ct between the transduced gene and <i>Gapdh</i>. The transduced mouse with the lowest expression of C/EBP-ε was used as calibrator. (H) Western blotting of C/EBP-ε, 24p3, and beta-actin in transduced cells from two mice. (I–J) Cells were fixed in formaldehyde. Cell and nuclear membranes were lysed before fragmentation of DNA by sonication. Chromatin was immunoprecipitated using protein A/G magnetic beads and an antibody against C/EBP-ε, C/EBP-α, or negative control rabbit IgG. After washing procedures, immune complexes were eluted and reversed and DNA recovered. DNA was used as a template for quantitative PCR. Primers used were specific for putative C/EBP sites in the <i>DEFA1</i> promoter and promoters of the specific granule protein cathelin-related antimicrobial peptide (<i>Camp</i>). Levels are depicted as fold enrichment compared to negative control IgG immunoprecipitation.</p

Chromatin immunoprecipitation (ChIP) analysis of the HNP-1 promoter in murine and human bone marrow (BM) cells.

<p>BM cells from 3 mice (A–B) and two healthy human donors (C–D) were extracted and fixed in formaldehyde. Cell and nuclear membranes were lysed before fragmentation of DNA by sonication. Chromatin was immunoprecipitated using protein A/G magnetic beads and an antibody against C/EBP-ε, C/EBP-α, or negative control rabbit IgG. After washing procedures, immune complexes were eluted and reversed and DNA recovered. DNA was used as a template for quantitative PCR. Primers used were specific for putative C/EBP sites in the (A,C) HNP-1 promoter and (B,D) murine and human promoters of the specific granule protein cathelin-related antimicrobial peptide (CAMP). Levels are depicted as fold enrichment compared to negative control IgG immunoprecipitation. Error bars depict standard deviation.</p